病理学与病理生理学 9no[1].10

单击此处编辑母版标题样式单击此处编辑母版副标题样式* 1eNOS and Cardiovascular diseases l Yong Jil Atherosclerosis Research Center l NanJing Medical University2008-11-20SUMMARYl The regulation of eNOS l eNOS-derived NO and atherosclerosisl Recent achievement单击此处编辑母版标题样式单击此处编辑母版副标题样式* 3The regulation of eNOSL-arginine NO + L-citrullineeNOScalmodulin (CaM) tetrahydrobiopterin(H4B)flavin mononucleotideFAD NADPHRegulation of eNOS expressionl Regulation of eNOS mRNA stabilityeNOS levels in endothelial cells can also be efficiently regulated by changes in eNOS mRNA stability.Hypoxia and cytokines,such as TNF,also downregulate eNOS mRNA levels by decreasing thehalf-life of eNOS mRNA from 48 h under basal conditions to 3 h.Intracellular Translocationl Functional eNOS can be detected in at least threemembrane compartments, the plasmamembrane, plasmalemmal caveolae, and theGolgi apparatus .l However, the disruption of the Golgi apparatus does not affect the agonist-induced, endothelium-dependent, and NO-mediated relaxation of porcinecoronary arteries, suggesting that the Golgi eNOS pool contributes little to the regulation of vasculartone.l Two groups have reported that eNOS can also bedetected in the nucleus of endothelial cells andbrown adipocytes . l Although the nuclear localization of eNOS mayfacilitate the interaction of NO with transcription factors, the conditions associated with a localization of eNOS and its exact role in thiscell compartment remain to be determined.eNOS signaling complexSignal transduction moleculesproteinsColocalizationEnzyme activaion NO production Downstream effector pathwayCa2+ and eNOS Activityl Ca2+-dependent activation of eNOSCaM binding to the CaM-binding motif is thought todisplace an adjacent autoinhibitory loop on eNOS (andnNOS),thus facilitating NADPH-dependent electronflux from the reductase domain of the protein to theoxygenase domain. (acetylcholine , bradykinin , histamine )l Ca2+-independent activation of eNOSfluid shearstress Activation of PI3KActivation of the serine kinases AktActivation of protein kinases AP-eNOSeNOSeNOS Phosphorylationl Ser 1177.When Ser1177 is phosphorylated,the flux of electrons through the reductase domain and,as a consequence, also NO production are increased two- to threefoldabove basal levels .l Thr495. This residue is constitutively phosphorylated in all of the endothelial cells investigated to date and is a negative regulatory site, phosphorylation is associated with a decrease in enzyme activity.Other Phosphorylation SitesSer633 Ser615 Ser114Tyr83Phosphorylation eNOS activity单击此处编辑母版标题样式单击此处编辑母版副标题样式* 13eNOS-derived NO and atherosclerosisAtherosclerosisThe Nobel Prize in Physiology or Medicine 1998Robert F. Furchgott Louis J. IgnarroFerid MuradFor their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular systemDiscovery of NOUncoupled eNOSeNOS uncoupling: electrons flowing from the reductase domain to the heme are diverted to molecular oxygen ratherthan to the substrate L-arginine, thereby resulting in production of superoxide instead of NO.The signal transduction pathways Promote vasorelaxationPrevent platelet aggregation and leukocytes adhesionInhibit vascular permeabilityInhibit atherogenesisL-arginineL-citrullineeNOSO2 NADPHNONADPEffect of eNOS-Derived NO on atherosclerosisInhibit vascular SMC migration ,proliferation单击此处编辑母版标题样式单击此处编辑母版副标题样式* 20Recent achievementTo investigate the mechanism by which this association between eNOS and -actin can regulate eNOS activity.-actin coimmunoprecipitates with eNOS in platelets.Ji Y, et al. Proc Natl Acad Sci U S A 2007;104:8839-8844. eNOS binds to the globular, but not the filamentous, form of -actin in platelets.Polymerization status of -actin regulates eNOS activity and NO bioactivity in platelets.Polymerization status of -actin has no change in intracellular Ca2+ or serine-1177 phosphorylation of eNOS.Hsp90 colocalizes with eNOS and -actin.-Actin, Hsp90, and eNOS were each detected (AC, respectively) by ImmunoGold labeling with electron microscopy. Triple labeling shows multiple areas (arrowed) where -actin (5-nm gold particles), eNOS(10-nm gold particles), and Hsp90 (15-nm gold particles) colocalize (D). g, platelet granule.Globular -actin forms a ternary complex with eNOS and Hsp90.(A) eNOS binding to monomeric -actin was concentration-dependent, and reversible.(B) Preincubation of eNOS with Hsp90 increased the degree of binding to immobilized monomeric -actin, by 2.81.2-fold, and this binding became irreversible.Conclusion l Here, we demonstrate in human platelets that the polymerization state of -actin crucially regulates the activation state of eNOS, and hence NO formation, through altering its binding of Hsp90.Articles in recent yearsl Ji Y, Ferracci G, Ferro A,et al. beta-Actin regulates platelet nitric oxide synthase 3 activity through interaction with heat shock protein 90. Proc Natl Acad Sci U S A 2007;104:8839-8844 l Chen L, Liu Y, Ji Y, et al. 17Beta-oestradiol partially attenuates the inhibition of nitric oxide synthase-3 by advanced glycation end-products in human platelets. Clin Exp Pharmacol Physiol 2007 Oct;34(10):972-8. l Queen LR, Ji Y, Ferro A, et al. Mechanisms underlying beta2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells. J Physiol 2006;576(pt2):585-94l Ji Y, Diao J, Han Y, et al. Pyridoxine prevents dysfunction of endothelial cell nitric oxide production in response to low-density lipoprotein. Atherosclerosis 2006; 188(1):84-94 l Biao Xu, Yong Ji, Kang Yao, et al. Inhibition of human endothelial cell nitric oxide synthesis by advanced glycation end-products but not glucose: relevance to diabetes. Clin Sci 2005;109:439-46. Articles in recent yearsl Ji Y, Han Y, Diao J, et al. Inhibition of endothelial nitric oxide generation by low-density lipoprotein is partially prevented by L-arginine and L-ascorbate. Atherosclerosis 2004;176:345-53.l Ferro A, Coash M, Ji Y, et al. Nitric oxide-dependent beta2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt. Br J Pharmacol 2004;143:397-403.l LR Queen,